scholarly journals Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

2017 ◽  
Vol 114 (32) ◽  
pp. 8614-8619 ◽  
Author(s):  
Joyce K. Hwang ◽  
Chong Wang ◽  
Zhou Du ◽  
Robin M. Meyers ◽  
Thomas B. Kepler ◽  
...  
Keyword(s):  
B Cells ◽  
Neutralizing Antibodies ◽  
Somatic Hypermutation ◽  
Human Subjects ◽  
Variable Region ◽  
Affinity Maturation ◽  
Broadly Neutralizing Antibodies ◽  
Variable Regions ◽  
Complementarity Determining Regions ◽  
Hiv 1

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.

scholarly journals Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models

2020 ◽  
Vol 117 (14) ◽  
pp. 7929-7940
Author(s):  
Ming Tian ◽  
Kelly McGovern ◽  
Hwei-Ling Cheng ◽  
Peyton Waddicor ◽  
Lisa Rieble ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mouse Models ◽  
Negative Selection ◽  
Cell Development ◽  
B Cell Development ◽  
Affinity Maturation ◽  
Variable Regions ◽  
Conditional Expression ◽  
Hiv 1

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1–infected individuals, bnAbs evolved from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar antibody maturation process. One way to test a vaccine is to immunize mouse models that express human bnAb precursors and assess whether the vaccine can convert precursor antibodies into bnAbs. A major problem with such mouse models is that bnAb expression often hinders B cell development. Such developmental blocks may be attributed to the unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are usually under negative selection during primary B cell development. To address this problem, we devised a method to circumvent such B cell developmental blocks by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of the VRC26UCA led to developmental arrest of B cell progenitors in bone marrow; poly-reactivity of the VRC26UCA and poor pairing of the VRC26UCA heavy chain with the mouse surrogate light chain may contribute to this phenotype. The conditional expression strategy bypassed the impediment to VRC26UCA B cell development, enabling the expression of VRC26UCA in mature B cells. This approach should be generally applicable for expressing other bnAbs that are under negative selection during B cell development.

scholarly journals Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies

Structure ◽  
2016 ◽  
Vol 24 (8) ◽  
pp. 1346-1357 ◽  
Author(s):  
Thaddeus M. Davenport ◽  
Jason Gorman ◽  
M. Gordon Joyce ◽  
Tongqing Zhou ◽  
Cinque Soto ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Somatic Hypermutation ◽  
Broadly Neutralizing Antibodies ◽  
Structure And Dynamics ◽  
Induced Changes ◽  
Hiv 1

scholarly journals Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Deniz Cizmeci ◽  
Giuseppe Lofano ◽  
Evan Rossignol ◽  
Anne-Sophie Dugast ◽  
Dongkyoon Kim ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Clonal Evolution ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Gene Repertoire ◽  
Immunoglobulin Gene ◽  
Hiv Controllers ◽  
Hiv 1

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

scholarly journals Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Victor Ovchinnikov ◽  
Joy E Louveau ◽  
John P Barton ◽  
Martin Karplus ◽  
Arup K Chakraborty
Keyword(s):  
B Cells ◽  
Neutralizing Antibodies ◽  
Affinity Maturation ◽  
Surface Proteins ◽  
Initial Increase ◽  
Broadly Neutralizing Antibodies ◽  
Rigidity Result ◽  
Conserved Residues ◽  
Immunogen Design ◽  
Dynamics Simulations

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.

scholarly journals Highly sensitive and unbiased approach for elucidating antibody repertoires

2016 ◽  
Vol 113 (28) ◽  
pp. 7846-7851 ◽  
Author(s):  
Sherry G. Lin ◽  
Zhaoqing Ba ◽  
Zhou Du ◽  
Yu Zhang ◽  
Jiazhi Hu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Variable Region ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Variable Regions ◽  
Antibody Repertoires ◽  
B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

scholarly journals Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen binding affinity

2018 ◽  
Author(s):  
Pia Dosenovic ◽  
Ervin E. Kara ◽  
Anna-Klara Pettersson ◽  
Andrew McGuire ◽  
Matthew Gray ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Neutralizing Antibodies ◽  
Cell Receptor ◽  
Broadly Neutralizing Antibodies ◽  
Precursor Frequency ◽  
The Relationship ◽  
Anti Hiv ◽  
Hiv 1

AbstractThe discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination, and instead that it would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain super-physiologic numbers of bNAb precursor expressing B cells and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center B cell recrutiment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the germinal center when there are as few as 10 such cells per mouse. However, at low precursor frequencies the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to germinal centers is variable and dependent on re-circulation.Significance statementAn essential requirement for an HIV-vaccine is to elicit antibodies to conserved regions of the spike protein (Env) becasue these antibodies can protect against infection. Although broadly neutralizing antibodies develop naturally in rare individuals after prolongued HIV infection, eliciting them by vaccination has only been possible in artificial knock-in mouse models wherein the number of B cells expressing the antibody precursor is super-physiologic. To understand the relationship between precursor frequency, antigen affinity and germinal center recruitment we have performed adoptive transfer experiments in which fixed numbers of precursor cells are engrafted in wild type mice. Our results provide a framework for understanding how precursor frequency and antigen affinity shape humoral immunity to HIV.

scholarly journals Enhancement of antibody-dependent cellular cytotoxicity and phagocytosis in anti‐HIV‐1 bovine chimeric broadly-neutralizing antibodies.

2021 ◽  
Author(s):  
Jack M Edwards ◽  
Behnaz Heydarchi ◽  
Georges Khoury ◽  
Natalia A Salazar-Quiroz ◽  
Christopher A Gonelli ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Variable Region ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Broadly Neutralizing Antibodies ◽  
Effector Functions ◽  
Human Igg1 ◽  
Topical Microbicides ◽  
Anti Hiv ◽  
Hiv 1

No prophylactic vaccine has provided robust protection against HIV-1. Vaccine-induced broadly neutralizing antibodies (bnAbs) have not been achieved in humans and most animals, however cows vaccinated with HIV-1 envelope trimers produce bnAbs with unusually long third heavy complementarity determining regions (CDRH3). Alongside neutralization, Fc-mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) may be critical for in vivo bnAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bnAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bnAbs by using an exceptionally long 60aa CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding: G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human NK cell activation and mediated higher levels of ADCC and ADP activity compared to the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity compared to GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultra-long CDRH3 regions could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, HIV is still a lifelong persistent virus and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 bnAbs and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bnAbs (with higher potency and breadth than most human bnAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high potency bovine variable region bnAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

The role of HIRA-dependent H3.3 deposition and its modifications in the somatic hypermutation of immunoglobulin variable regions

2021 ◽  
Vol 118 (50) ◽  
pp. e2114743118
Author(s):  
Guojun Yu ◽  
Yongwei Zhang ◽  
Varun Gupta ◽  
Jinghang Zhang ◽  
Thomas MacCarthy ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Variable Region ◽  
Chromatin Accessibility ◽  
Variable Regions ◽  
Igh Locus ◽  
Human B Cells ◽  
Active Transcription ◽  
Activation Induced Deaminase

The H3.3 histone variant and its chaperone HIRA are involved in active transcription, but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells are not yet fully understood. In this study, we show that the knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of the immunoglobulin heavy chain (IgH) in the human Ramos B cell line without changing the levels of activation-induced deaminase and other major proteins known to be involved in SHM. Except for H3K79me2/3 and Spt5, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells, and this was accompanied by decreased nascent transcription in the IgH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the IgH locus was also reduced in the HIRA KO. Somewhat surprisingly, HIRA loss increased the chromatin accessibility of the IgH V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with the HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IgH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription-associated chromatin structure and accessibility contribute to SHM in human B cells.

scholarly journals How Germinal Centers Evolve Broadly Neutralizing Antibodies: the Breadth of the Follicular Helper T Cell Response

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Rob J. De Boer ◽  
Alan S. Perelson
Keyword(s):  
B Cells ◽  
Neutralizing Antibodies ◽  
Evolutionary Process ◽  
Germinal Centers ◽  
Broadly Neutralizing Antibodies ◽  
Cell Repertoire ◽  
Tfh Cells ◽  
Follicular Helper ◽  
Neutralizing Capacity ◽  
Hiv 1

ABSTRACT Many HIV-1-infected patients evolve broadly neutralizing antibodies (bnAbs). This evolutionary process typically takes several years and is poorly understood as selection taking place in germinal centers occurs on the basis of antibody affinity. B cells with the highest-affinity receptors tend to acquire the most antigen from the follicular dendritic cell (FDC) network and present the highest density of cognate peptides to follicular helper T (Tfh) cells, which provide survival signals to the B cell. bnAbs are therefore expected to evolve only when the B cell lineage evolving breadth is consistently capturing and presenting more peptides to Tfh cells than other lineages of more specific B cells. Here we develop mathematical models of Tfh cells in germinal centers to explicitly define the mechanisms of selection in this complex evolutionary process. Our results suggest that broadly reactive B cells presenting a high density of peptides bound to major histocompatibility complex class II molecules (pMHC) are readily outcompeted by B cells responding to lineages of HIV-1 that transiently dominate the within host viral population. Conversely, if broadly reactive B cells acquire a large variety of several HIV-1 proteins from the FDC network and present a high diversity of several pMHC, they can be rescued by a large fraction of the Tfh cell repertoire in the germinal center. Under such circumstances the evolution of bnAbs is much more consistent. Increasing either the magnitude of the Tfh cell response or the breadth of the Tfh cell repertoire markedly facilitates the evolution of bnAbs. Because both the magnitude and breadth can be increased by vaccination with several HIV-1 proteins, this calls for experimental testing. IMPORTANCE Many HIV-infected patients slowly evolve antibodies that can neutralize a large variety of viruses. Such broadly neutralizing antibodies (bnAbs) could in the future become therapeutic agents. bnAbs appear very late, and patients are typically not protected by them. At the moment, we fail to understand why this takes so long and how the immune system selects for broadly neutralizing capacity. Typically, antibodies are selected based on affinity and not on breadth. We developed mathematical models to study two different mechanisms by which the immune system can select for broadly neutralizing capacity. One of these is based upon the repertoire of different follicular helper T (Tfh) cells in germinal centers. We suggest that broadly reactive B cells may interact with a larger fraction of this repertoire and demonstrate that this would select for bnAbs. Intriguingly, this suggests that broadening the Tfh cell repertoire by vaccination may speed up the evolution of bnAbs.

scholarly journals The germinal center antibody response in health and disease

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 999 ◽  
Author(s):  
Anthony L. DeFranco
Keyword(s):  
B Cells ◽  
Antibody Response ◽  
Germinal Center ◽  
Neutralizing Antibodies ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Effective Control ◽  
Immune Recognition ◽  
Immunoglobulin Gene ◽  
Variable Regions

The germinal center response is the delayed but sustained phase of the antibody response that is responsible for producing high-affinity antibodies of the IgG, IgA and/or IgE isotypes. B cells in the germinal center undergo re-iterative cycles of somatic hypermutation of immunoglobulin gene variable regions, clonal expansion, and Darwinian selection for cells expressing higher-affinity antibody variants. Alternatively, selected B cells can terminally differentiate into long-lived plasma cells or into a broad diversity of mutated memory B cells; the former secrete the improved antibodies to fight an infection and to provide continuing protection from re-infection, whereas the latter may jumpstart immune responses to subsequent infections with related but distinct infecting agents. Our understanding of the molecules involved in the germinal center reaction has been informed by studies of human immunodeficiency patients with selective defects in the production of antibodies. Recent studies have begun to reveal how innate immune recognition via Toll-like receptors can enhance the magnitude and selective properties of the germinal center, leading to more effective control of infection by a subset of viruses. Just as early insights into the nature of the germinal center found application in the development of the highly successful conjugate vaccines, more recent insights may find application in the current efforts to develop new generations of vaccines, including vaccines that can induce broadly protective neutralizing antibodies against influenza virus or HIV-1.

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